Peptides and compositions for prevention of cell adhesion and methods of using same

ABSTRACT

Compositions comprising an isolated peptide, which may for example optionally comprise a sequence selected from the group consisting of YDYNWY (SEQ ID NO: 1), YDYNLY (SEQ ID NO: 2), FDYNFY (SEQ ID NO: 3), FDYNLY (SEQ ID NO: 4), FDYNWY (SEQ ID NO: 5), YDWNLY (SEQ ID NO: 6), YDWHLY (SEQ ID NO: 7), and WDYNLY (SEQ ID NO: 8), extracted from organisms such as aquatic organisms and moss or any other sequence described herein, and methods of using same, including for treatment of or prevention of formation of microbial biofilms and against adhesion of a cell to a surface.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 14/708,014, filed May 8, 2015, which is a continuation of U.S.patent application Ser. No. 13/120,049, filed Mar. 21, 2011, now U.S.Pat. No. 9,029,318, which is a U.S. National Stage of PCT/IB2009/006926,filed Sep. 23, 2009, and claims priority of U.S. Provisional PatentApplication No. 61/136,673, filed Sep. 24, 2008. The disclosures of theprior applications are incorporated by reference herein in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 8, 2014, isnamed 095432-0125_SL.txt and is 38,421 bytes in size.

FIELD OF THE INVENTION

The present invention relates to isolated natural peptides and their usein prevention of cell adhesion.

BACKGROUND OF THE INVENTION

Microorganisms can live and proliferate as individual cells swimmingfreely in the environment (as plankton), or they can grow as highlyorganized, multicellular communities encased in a self-producedpolymeric matrix in close association with surfaces and interfaces. Thelatter microbial lifestyle is referred to as biofilms. Biofilm formationrepresents an ancient, protected mode of growth that allows microbialsurvival in hostile environments and allows microorganisms to disperseand colonize new niches [Hall-Stoodley et al., Nat Rev Microbiol. (2004)2(2):95-108].

The composition of biofilms is complex and variable among differentmicrobial species and even within the same species under differentenvironmental conditions. Nonetheless, biofilm formation represents thenormal lifestyle of microorganism in the environment and all microbescan make biofilms. Previous studies revealed that bacterial biofilmformation progresses through multiple developmental stages differing inprotein profiles [Sauer et al., J Bacteriol. (2002) 184(4):1140-54],beginning with attachment to surface, followed by the immigration anddivision to form microcolonies and finally maturation involvingexpression of matrix polymers. Bacteria within each biofilm stagedisplay phenotypes and possess properties that are markedly differentfrom those of the same group growing planktonically [Sauer et al., JBacteriol. (2004) 186(21):7312-26].

Biofilms are a major cause of systemic infections (e.g. nosocomialinfections) in humans. In the body, biofilms can be associated withtissues (e.g., inner ears, teeth, gums, lungs, heart valves and theurogenital tract). An estimated 65% of bacterial infections in humansare biofilm in nature. Additionally, after forming biofilms,microorganisms tend to change their characteristics, sometimesdrastically, such that doses of antibiotics which normally kill theorganisms in suspended cultures are completely ineffective against thesame microorganisms when the organisms are in attached or conglomeratebiofilm form (U.S. Pat. No. 7,189,351).

One of the principal concerns with respect to products that areintroduced into the body (e.g., contact lenses, central venouscatheters, mechanical heart valves and pacemakers) or provide a pathwayinto the body is microbial infection and invariably biofilm formation.As these infections are difficult to treat with antibiotics, removal ofthe device is often necessitated, which is traumatic to the patient andincreases the medical cost. Accordingly, for such medical apparatuses,the art has long sought means and methods of rendering those medicalapparatuses and devices antimicrobial.

PCT Application No. WO 06/006172 discloses the use of anti-amyloidagents, such as aromatic compounds, for inhibiting formation ordisintegrating a pre existing biofilm. The application discloses thatcompounds preventing amyloid fibril formation in Alzheimer can actagainst fibril formation in biofilms, and concludes that amino acidshaving an aromatic arm are effective against biofilms. However, theanalysis was limited to full length sequences.

SUMMARY OF THE INVENTION

The present invention provides broad spectrum natural factors thatinterfere with biofilm formation at its initial stages, in a wide rangeof microorganisms. From these natural factors, peptides with highconservation sequences were isolated, and showed high activity inprevention of microbial adherence in its synthetic conformation. Theconserved sequence is found in several marine organisms, includingvarious known species of sea anemone, several fish (including Daniorerio—zebra fish), and in moss Physcomitrella patens subsp. patens.

All factors mentioned above show activity that is exclusively directedto the prevention of bacterial substrate adhesion and the derivedbiofilm formation. It is devoid of the commonly observed lethalbactericidal activity, revealed by the antibiotic peptides and secondarymetabolites, which provides a strong selective pressure for rapidnatural selection by the intensive microbial “biotic potential.” On theother hand a wide range inhibition of bacterial colonization antagonizesa fundamental mechanism of bacterial survival. Therefore an adaptivemodification of such mechanism has a low likelihood due to its vitality.

Sher et al. (Toxicon 45: 865-879, 2005) identified putative biologicallyactive proteins and polypeptides expressed by hydrae which could becomponents of its allomonal system, using a bioinformatics approach.Hydrae were shown to express orthologs of cnidarian phospholipase A2toxins and cytolysicns belonging to the actinoporin family, and toexpress proteins similar to elapid-like phospholipases, cysteine-richsecretory proteins (CRISP), prokineticin-like polypeptides and toxicdeoxyribonucleases.

The specific sequences responsible for cytotoxic activity in peptidesisolated from natural sources have not hitherto been identified.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

As used herein, the terms “comprising” and “including” or grammaticalvariants thereof are to be taken as specifying the stated features,integers, steps or components but do not preclude the addition of one ormore additional features, integers, steps, components or groups thereof.This term encompasses the terms “consisting of” and “consistingessentially of.”

The phrase “consisting essentially of” or grammatical variants thereofwhen used herein are to be taken as specifying the stated features,integers, steps or components but do not preclude the addition of one ormore additional features, integers, steps, components or groups thereofbut only if the additional features, integers, steps, components orgroups thereof do not materially alter the basic and novelcharacteristics of the claimed composition, device or method. The term“method” refers to manners, means, techniques and procedures foraccomplishing a given task including, but not limited to, those manners,means, techniques and procedures either known to, or readily developedfrom known manners, means, techniques and procedures by practitioners ofthe chemical, biological and biophysical arts.

As used herein the term “about” refers to ±10%.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 shows the crystal structure of the 1GWY chain A of thewater-soluble state of the pore-forming cytolysin sticholysin Ii, theactive region marked in yellow; (and circled)

FIG. 2 shows the crystal structure of the 1GWY chain B of thewater-soluble state of the pore-forming cytolysin sticholysin Ii, theactive region marked in yellow; (and circled)

FIG. 3 shows the structure of the IKD6 chain A of the eukaryoticpore-forming cytolysin equinatoxin Ii, the active region marked inyellow; (and circled)

FIG. 4 shows the 3-dimensional construct of an equinatoxin mutant, theactive region marked in yellow; (and circled)

FIG. 5 is a bar chart showing the effects of different concentrations ofsynthetic proteins on growth of Pseudomonas aeruginosa ATee 27853 over24 hours; No bactericidal or bacteriostatic effect;

FIG. 6 is a bar chart showing the effects of different concentrations ofsynthetic proteins on biofilm formation by Pseudomonas aeruginosa ATee27853 over 24 hours;

FIG. 7 is a bar chart showing the effects of different concentrations ofsynthetic proteins on growth of a clinical isolate of AcinetobacterBaumannii over 24 hours; No bactericidal or bacteriostatic effect;

FIG. 8 is a bar chart showing the effects of different concentrations ofsynthetic proteins on biofilm formation by a clinical isolate ofAcinetobacter Baumannii over 24 hours;

FIG. 9 is a bar chart showing the effects of selected tentacularfractions from Actinia equina on biofilm formation by Acinetobacterbaumannii, with PBS as a positive control;

FIG. 10 is a bar chart showing the effects of fraction 13 on biofilmformation by various gram positive and gram negative bacteris;

FIG. 11 is a bar chart showing the effects of crude extracts fromAnemonia, Aiptasia and Physcomitrella (Moss) on Pseudomonas aeruginosain protein concentration of 50 μg/ml;

FIG. 12 is a bar chart showing the effects of five synthetic peptidesand crude material from the moss of Physcomitrella patens;

FIG. 13 shows fractions obtained by separation of crude extract ofAiptasia pulchella on Sephadex G-10 column;

FIG. 14 shows peaks obtained by rechromatography of the high molecularweight fraction of FIG. 13;

FIG. 15 shows fractions obtained by reversed phase high performanceliquid chromatography (RP-HPLC) separation with c-18 column, of the lowmolecular fraction of FIG. 14;

FIGS. 16A-B show the general structure of a cyclic lead with emulsifierarm of a peptide according to the principles of the present invention;and

FIG. 17 is a flow chart showing development of a cyclic peptide leadwith emulsifying arm.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of compositions comprising an isolated peptidewhich has one or more effects relating to prevention of bacterialsubstrate adhesion and the derived biofilm formation, and optionallyalso prevention of cell-cell adhesion. Other effects may also optionallybe provided, additionally or alternatively. As a non-limiting example,the peptide may optionally and preferably comprise a sequence selectedfrom the group consisting of YDYNWY (SEQ ID NO: 1), YDYNLY (SEQ ID NO:2), FDYNFY (SEQ ID NO: 3), FDYNLY (SEQ ID NO: 4), FDYNWY (SEQ ID NO: 5),YDWNLY (SEQ ID NO: 6), YDWHLY (SEQ ID NO: 7) and WDYNLY (SEQ ID NO: 8),extracted from organisms such as aquatic organisms and moss and methodsof using same. Other sequences are described below.

One of the major concerns in medicine is microbial biofilm formation. Inhumans, biofilms are a cause of systemic infections (e.g., nosocomialinfections) and are a major concern when introducing products into thebody (e.g., contact lenses, central venous catheters, mechanical heartvalves and pacemakers).

Biofilms are also a problem in many industries including the food,pharmaceutical, paint, water, shipping and engineering industriescausing, amongst a wide range of negative effects, accelerated corrosionin industrial systems, oil souring and biofouling. For example,biofouling may be caused by the adhesion of organisms to any surface ina marine or freshwater environment, including cooling towers, waterpipes and filters in cooling or desalinization installations, irrigationand power stations, and membranes, such as those used in wastewater anddesalinization systems. Biofouling also occurs in aquaculture systems infish farms.

Furthermore the commercial shipping fleets of the world consumeapproximately 300 million tones of fuel annually. Without antifoulingmeasures, that fuel consumption would increase by as much as 40%,equivalent to an extra 120 million tones of fuel annually. The economiccost of this was estimated as about $7.5 billion in 2000; a more recentestimate is $30 billion.

Biofilms are very difficult to eliminate Since microbes growing withinare highly organized and can withstand hostile environments, such ashigh temperatures and anti-microbial agents (e.g., antibiotics).

Marine and fresh water plants and organisms including soft bodied waterinvertebrates, fish and moss are surrounded by broad spectrum species ofmicrobial organisms. Since such plant and organisms lack specificimmunity, they produce several factors which can prevent microbialcolonization on their body surface.

The most “sensitive” organisms are invertebrates belong to the phylumcnidaria that include the sea anemones, corals, jellyfish, hydroids,medusae, and sea fans. Such soft bodied organism, which lack physicalprotection such as scales or shells, use proteins and secondarymetabolites to protect themselves from the microbial environmentsurrounding them.

It has been previously reported that marine organisms (e.g. sponges)produce secondary metabolites that exhibit antibacterial and antifungalactivities [Amade et al., supra). Moreover, sea anemones (e.g., Actiniaequina) have been shown to produce toxic, pore forming peptides (i.e.,equinatoxins), which lyse and kill eukaryotic cells similarly to othersmall antimicrobial peptides [Anderluh et al., supra).

Although it is known in the art that the full length sequences ofvarious proteins are related to their cytolysic function, the specificpeptides responsible for the cytolysic effect have not been previouslyidentified.

The present inventors have demonstrated that several active fractionsobtained from sea anemones using liquid chromatography separations showa high level of prevention of microbial adherence to abiotic surfaces.The sea anemone includes 46 families that can be found in water sourcesaround the world. Most sea anemones are sessile, with a specialized footused to anchor them in soft substrates, or attach themselves to rocksand corals. The anti-adhesive activity was demonstrated with severalspecies of sea anemone belonging to different genera: Actinia equine,Aiptasia and Anemonia. The N terminus region of anemone cytotoxin hasbeen shown to be involved in the cytotoxic effect [Ref: Kristan K,Podlesek Z, Hojnik V, Gutierrez-Aguirre I, Gunčar G, Turk D,Gonzalez-Manas J M, Lakey J H, Maček P, Anderluh G (2004): Poreformation by equinatoxin, an eukaryotic pore-forming toxin, requires aflexible N-terminal region and a stable beta sandwich, J Biol Chem,279(45):46509-46517]. A protein having some resemblance to the Cterminus region of anemone cytotoxin, which region is not involved incytotoxicity, has also been identified in fish by the present inventors.This protein has a highly conserved region, with unknown function, whichis also a Trp-rich domain, and may be important for binding of theprotein to lipid membrane. The present inventors have also found thisregion in the moss Physcomitrella patens.

The present inventors therefore hypothesized that this region provides apeptide which is highly effective in prevention in biofilm formation,while being devoid of cytotoxic activity. The present inventors havecharacterized and isolated a natural peptide comprising a sequenceselected from the group consisting of YDYNWY (SEQ ID NO: 1), YDYNLY (SEQID NO: 2), FDYNFY (SEQ ID NO: 3), FDYNLY (SEQ ID NO: 4), WDYNLY (SEQ IDNO: 8), FDYNWY (SEQ ID NO: 5), YDWNLY (SEQ ID NO: 6) and YDWHLY (SEQ IDNO: 7), having highly effective anti-biofilm properties.

According to some embodiments, the peptide comprises part of a sequencecomprising 10 up to about 30, up to about 40, or up to about 50 aminoacids.

According to some embodiments, the peptide is selected from the groupconsisting of LFSVPYDYNWYSNWW (SEQ ID NO: 9), FSVPYDYNLYSNWW (SEQ ID NO:10), MFSVPFDYNFYSNWW (SEQ ID NO: 11), MFSVPFDYNLYSNWW (SEQ ID NO: 12),MFSVPFDYNLYTNWW (SEQ ID NO: 13), MWSVPFDYNLYSNWW (SEQ ID NO: 14),MFSVPWDYNLYKNWF (SEQ ID NO: 15), MFSVPFDYNLYKNWL (SEQ ID NO: 16),MFSVPFFDYNWYSNWW (SEQ ID NO: 17), LFSVPFDYNLYSNWW (SEQ ID NO: 18),LFSVPYDYNWYSNWW (SEQ ID NO: 9), MASIPYDWNLYQSWA (SEQ ID NO: 19),MASIPYDWNLYSAWA (SEQ ID NO: 20), and MASIPYDWHLYNAWA (SEQ ID NO: 21). Asis shown herein below and in the Examples section which follows, thepresent inventors have identified an active fraction extracted fromAiptesia pulchella anemone, using tandem mass spectroscopy (MS/MS)analysis.

The present inventors used the clustaLW program to identify biologicallymeaningful 20 multiple sequence alignments of several anemone cytotoxinproteins and identify an anemone cytotoxin universal primer for use in apolymerase chain reaction (PCR). Amplification of a 250 bp region ofcytotoxin proteins from two different anemones, Aiptesia and Anemoniaviridansm, of sequence Eqt-F: GTR TCG ACA ACG AGT CRG G (SEQ ID NO: 22)and Eqt-R252: TGA CAT YCC ACC AGT TGC TG (SEQ ID NO: 23), respectively,was achieved. Translation of these regions to peptides, and BlastXcomparison to the genebank, showed that these regions are part of theconserved domain of anemone cytotoxin. As discussed in greater detail inthe Examples section below, and shown in FIGS. 5 to 8, the presentinventors compared the activities of a number of synthetic peptides fromanemones and moss, and found that these peptides prevented the formationof biofilm [FIGS. 6 and 8 to 12], but did not kill or inhibit growth ofbacteria [FIGS. 5 and 7].

The anti-adhesive effect was demonstrated on several bacterial species(FIG. 10), which led the present inventors to conclude that the activematerials are not species specific but active against a broad range ofmicrobial species.

The conserved peptide region has been identified, for example, in thefollowing natural proteins: (SEQ ID NOS 24-31, respectively, in order ofappearance)

LFSVPYDYNWYSNWW EqT-IV FSVPYDYNLYSNWW Actinoporin Or-A MFSVPFDYNFYSNWWHMg III from Heteractis magnifica MFSVPFDYNLYSNWW Avt-I RTX-AMFSVPFDYNLYTNWW Pstx20 MWSVPFDYNLYSNWW Physcomitrella patensMFSVPWDYNLYKNWF Danio rerio MFSVPFDYNLYKNWL Tetraodon nigroviridis

Optionally and preferably, the peptide of the present inventioncomprises the sequence CNIFSVPFDYNWYSNWWC (SEQ ID NO: 32). Optionallyand preferably, the peptide of the present invention is comprised in aprotein having from about 100 to about 300 amino acids.

Without wishing to be limited by a single hypothesis, based on the3-dimensional structure of 2 anemone cytotoxin (equinatoxin andSticholysin), as shown in FIGS. 1-4, the active region faces outwards.

FIGS. 1 and 2 shows the crystal structure of the 1GWY chains A and B,respectively, of cytolysin sticholysin Ii. FIG. 3 shows the structure ofthe 1KD6 chain A of the eukaryotic pore-forming cytolysin equinatoxin

FIG. 4 demonstrates the 3-dimensional construct of an equinatoxinmutant, having three cysteines introduced at positions 8, 18 and 69(1TZQ Chain A). This mutant has been previously shown not to behemolytically active (Kristan K, Podlesek Z, Hojnik V, Gutierrez-AguirreI, Guncar G, Turk D, Gonzalez-Manas J M, Lakey J H, Macek P, Anderluh G(2004): Pore formation by equinatoxin, an eukaryotic pore-forming toxin,requires a flexible N-terminal region and a stable beta sandwich, J BiolChem. 279(45):46509-46517). The protein thus lost its cytotoxicity, butwas still active against bacterial adherence.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

According to one aspect of the present invention, there is provided acomposition comprising an isolated natural peptide, the peptidecomprising a sequence selected from the group consisting of YDYNWY (SEQID NO: 1), YDYNLY (SEQ ID NO: 2), FDYNFY (SEQ ID NO: 3), FDYNLY (SEQ IDNO: 4), WDYNLY (SEQ ID NO: 8), FDYNWY (SEQ ID NO: 5), YDWNLY (SEQ ID NO:6) and YDWHLY (SEQ ID NO: 7).

According to an additional aspect of the present invention there isprovided a method of preventing adhesion of a single cell organism to asurface, the method comprising contacting the cell with a composition ofcomprising an isolated natural peptide comprising a sequence selectedfrom the group consisting of YDYNWY (SEQ ID NO: 1), YDYNLY (SEQ ID NO:2), FDYNFY (SEQ ID NO: 3), FDYNLY (SEQ ID NO: 4), WDYNLY (SEQ ID NO: 8),FDYNWY (SEQ ID NO: 5), YDWNLY (SEQ ID NO: 6) and YDWHLY (SEQ ID NO: 7),thereby preventing adhesion of a cell to a surface.

According to some embodiments of the present invention, there ispreferably provided a domain which comprises at least one of the abovepeptides and which is effective against cell adhesion to a surface. Morepreferably, the domain is included as part of a protein. Optionally andmost preferably, the domain exhibits anti-adhesive behavior, for examplefor the prevention of formation and/or treatment of a biofilm, but doesnot exhibit cytotoxic behavior.

A non-limiting selection of exemplary domains is provided in the tablebelow.

Domain sequence Species LFSVPYDYNWYSNWW EqT-IV (SEQ ID NO: 24)FSVPYDYNLYSNWW Actinoporin Or-A (SEQ ID NO: 25) MFSVPFDYNFYSNWWHMg III from Heteractis magnifica (SEQ ID NO: 26) MFSVPFDYNLYSNWWAvt-I RTX-A (SEQ ID NO: 27) MFSVPFDYNLYTNWW Pstx20 (SEQ ID NO: 28)MWSVPFOYNLYSNWW Physcomitrella patens (SEQ ID NO: 29) MFSVPWDYNLYKNWFDanio rerio (SEQ ID NO: 30) MFSVPFDYNLYKNWL Tetraodon nigroviridis(SEQ ID NO: 31)

Further exemplary sequences are described herein, as being related tothe following sequence:

(SEQ ID NO: 33) MSRLIIVFIVVTMICSATALPSKKIIDEDEEDEKRSADVAGAVIDGASLSFDILKTVLEALGNVKRKIAVGVDNESGKTWTALNTYFRSGTSDIVLPHKVPHGKALLYNGQKDRGPVATGAVGVLAYLMSDGNTLAVLFSVPYDYNWYSNWWNVRIYKGKRRADQRMYEELYYNLSPFRGDNGWHTRNLGYGLKSRGFMN SSGHAILEIHVSKA.

This sequence has the GenBank accessionidentifier: >gi|48428895|sp|P61914.1|ACTP2_ACTEQ Equinatoxin-2 precursor(Equinatoxin II) (EqT II) (EqTII) Actinia equine and is 214 aa inlength. This sequence is also optionally an exemplary sequence accordingto the present invention. Positions 38-213 of this sequence hit theannotated domain pfam06369, Anemone_cytotox, Sea anemone cytotoxicprotein; therefore, this portion of the above sequence is alsooptionally an exemplary sequence according to the present invention.

In some embodiments, the present invention also includes any relatedsequence to the above sequence thereof. Such related sequences mayoptionally be found by running any type of sequence comparison software,including but not limited to BLASTP. Below are provided representativehits from selected taxa and their alignments to EqtII (the abovesequence):

1. Sea Anemones—

1a. Stichodactyla helianthus

>gi|2815496|sp|PO7845.2|ACTP2_STOHE Sticholysin-2 (Sticholysin II)(StnII) (Cytolysin St II) (Cytolysin III) (Cytotoxin)

(SEQ ID NO: 34) ALAGTIIAGASLTFQVLDKVLEELGKVSRKIAVGIDNESGGTWTALNAYFRSGTTDVILPEFVPNTKALLYSGRKDTGPVATGAVAAFAYYMSSGNTLGVMFSVPFDYNWYSNWWDVKIYSGKRRADQGMYEDLYYGNPYRGDNGWHEKNLGYGLRMKGIMTSAGEAKMQIKISRAlignment:

>sp|PO7845.2|ACTP2_STOHE Sticholysin-2 (Sticholysin II) (StnII)(Cytolysin St II) (Cytolysin III) (Cytotoxin)

Length=175

Score=253 bits (646), Expect=8e-66, Method: Composition-based stats.

Identities=118/176 (67%), Positives=144/176 (81%), Gaps=1/176 (0%)

Query  38 DVAGAVIDGASLSFDILKTVLEALGNVKRKIAVGVDNESGKTWTALNTYFRSGTSDIVLP 97 +AG +I GASL+F +L VLE LG V RKIAVG+DNESG TWTALN YFRSGT+D++LP Sbjct   1ALAGTIIAGASLTFQVLDKVLEELGKVSRKIAVG1DNESGGTWTALNAYFRSGTTDVILP  60 Query 98 HKVPHGKALLYNGQKDRGPVATGAVGVLAYLMSDGNTLAVLFSVPYDYNWYSNWWNVRIY 157 VP+KALLY+G+KD GPVATGAV AY MS GNTL V+FSVP+DYNWYSNWW+V+IY Sbjct  61EFVPNTKALLYSGRKDTGPVATGAVAAFAYYMSSGNTLGVMFSVPFDYNWYSNWWDVKIY 120 Query158 KGKRRADQRMYEELYYNLSPFRGDNGWHTRNLGYGLKSRGFMNSSGHA1LE1HVSK 213GKRRADQ MYE+LYY +P+RGDNGWH +NLGYGL+ +G M S+G A ++1 +S+ Sbjct 121SGKRRADQGMYEDLYYG-NPYRGDNGWHEKNLGYGLRMKG1MTSAGEAKMQ1K1SR 175(The above “Query” and “Sbjt” sequences disclosed as SEQ ID NOS 35 and34, respectively.)2. Bony Fish2a. Danio rerio

>gi|125821212|ref|XP_001342650.1|PREDICTED: hypothetical protein [Daniorerio]

(SEQ ID NO: 36) MTESAEAVAANVSSRRHATVEITNLTNNYCFLNPKVYLENGETSNPPQPTVRPLKTEVCTFSKSAAHATGSVGVLTYDLFERRRNDYTETLAIMFSVPWDYNLYKNWFAVGIYPKGKECDQALYKEMYYQKNQHGFVREEANGSGINFEGKNLD1RATMCPMGRAIVKVEVWDKLLSPMAQMDCAlignment:

>ref|XP_001342650.1|UniGene infoGene info PREDICTED: hypotheticalprotein [Danio rerio]

Length=184

GENE ID: 100002992 apnl| actinoporin-like protein [Danio rerio]

Score=199 bits (505), Expect=1e-49, Method: Composition-based stats.

Identities=49/167 (29%), Positives=73/167 (43%), Gaps=12/167 (7%)

Query  58 LEALGNVKRKIAVGVDNESG-KTWTALNTYFRSGTSDIVLPHKVPHGKALLYNGQKDRGP116 + A + +R V + N + + Y +G + V K + K Sbjct   8VAANVSSRRHATVEITNLTNNYCFLNPKVYLENGETSNPPQPTVRPLKTEVCTFSKSAAH  67 Query117 VATGAVGVLAYLMSD------GNTLAVLFSVPYDYNWYSNWWNVRIYKGKRRADQRMYEE 170ATG+VGVL Y + + TLA++FSVP+DYN Y NW+ V IY + DQ +Y+E Sbjct  68ATGSVGVLTYDLFERRRNDYTETLAIMFSVPWDYNLYKNWFAVGIYPKGKECDQALYKE 126 Query171 LYYNLSPF----RGDNGWHTRNLGYGLKSRGFMNSSGHAILEIHVSK 213 +YY +NG G L R M G AI+++ V Sbjct 127MYYQKNQHGFVREEANGSGINFEGKNLDIRATMCPMGRAIVKVEVWD 173(The above “Query” and “Sbjct” sequences disclosed as SEQ ID NOS 37-38,respectively.)2b. Tetraodon Nigroviridis

>gi|47218822|emb|CAG02807.1| unnamed protein product [Tetraodonnigroviridis]

(SEQ ID NO: 39) MESAEAVAADVSRSRSVTIEISNLTKNYCLINPRVYLESGETYNPPQPTVRPLMTEVCTFSKSSGIPTGSVGVLTYELLERRSTMLPETLAIMFSVPYDYSFYNNWFAVGIYETGTKCNEGLYKQMYNEKKQAEHGFVREKANGSGINYVGGNLDIRATMNPLGKAIMKVEVWDAFFPFSEAlignment:

>emb|CAG02807.1| unnamed protein product [Tetraodon nigroviridis]

Length=181

Score=192 bits (489), Expect=1e-47, Method: Composition-based stats.

Identities=46/170 (27%), Positives=76/170 (44%), Gaps=14/170 (8%)

Query  58 LEALGNVKRKIAVGVDNES-GKTWTALNTYFRSGTSDIVLPHKVPHGKALLYNGQKDRGP116 + A + R + + + N + Y SG + V + K G Sbjct   7VAADVSRSRSVTIEISNLTKNYCLINPRVYLESGETYNPPQPTVRPLMTEVCTFSKSSG  65 Query117 VATGAVGVLAYLMSD------GNTLAVLFSVPYDYNWYSNWWNVRIYKGKRRADQRMYEE 170 +TG+VGVL Y + + TLA++FSVPYDY++Y+NW+ V IY+ + ++ +Y++ Sbjct  66IPTGSVGVLTYELLERRSTMLPETLAIMFSVPYDYSFYNNWFAVGIYETGTKCNEGLYKQ 125 Query171 LYYNLSPF------RGDNGWHTRNLGYGLKSRGFMNSSGHAILEIHVSKA 214+Y NG +G L R MN G AI+++ V A Sbjct 126MYNEKKQAEHGFVREKANGSGINYVGGNLD1RATMNPLGKAIMKVEVWDA 175(The above “Query” and “Sbjct” sequences disclosed as SEQ ID NOS 40-41,respectively.)3. Mosses3a. Physcomitrella Patens

>gi|168060237|ref|XP_001782104.1| predicted protein [Physcomitrellapatens subsp. patens]

(SEQ ID NO: 42) MVVHLIAMGLRYSETIMKTARMAEAIIPAAELSIKTLQNIVEGITGVDRKIAIGFKNLTDYTLENLGVYFNSGSSDRSIAYKINAQEALLFSARKSDHTARGTVGTFSYYIQDEDKTVHVMWSVPFDYNLYSNWWNIAVVDGRQPPDSNVHDNLYNGSGGMPYPNKPDQYINNEQKGFHLFGSMTNNG QATIEVELKKA

>ref|XP_001782104.1|Gene info predicted protein [Physcomitrella patenssubsp. patens]

gb|EDQ53098.1|Gene info predicted protein [Physcomitrella patens subsp.patens]

Length=199

GENE ID: 5945292 PHYPADRAFT_61094|hypothetical protein [Physcomitrellapatens subsp. patens]

Score=230 bits (586), Expect=7e-59, Method: Composition-based stats.

Identities=63/183 (34%), Positives=1011183 (55%), Gaps=4/183 (2%)

Query  35 RSADVAGAVIDGASLSFDILKTVLEALGNVKRKIAVGVDNESGKTWTALNTYFRSGTSDI 94 ++A +A A+I A LS L+ ++E + V RKIA+G N + T L YF SG+SD Sbjct  18KTARMAEAIIPAAELSIKTLQNIVEGITGVDRKIAIGFKNLTDYTLENLGVYFNSGSSDR  77 Query 95 VLPHKVPHGKALLYNGQKDRGPVATGAVGVLAYLMSD-GNTLAVLFSVPYDYNWYSNWWN 153 ++K+ +ALL++ +K A G VG +Y + D T+ V++SVP+DYN YSNWWN Sbjct  78SIAYKINAQEALLFSARKSDH-TARGTVGTFSYYIQDEDKTVHVMWSVPFDYNLYSNWWN 136 Query154 VRIYKGKRRADQRMYEELYYNL--SPFRGDNGWHTRNLGYGLKSRGFMNSSGHAILEIHV 211 + +G++ D +++ LY P+ + N G G M ++G A +E+ +   Sbjct 137IAVVDGRQPPDSNVHDNLYNGSGGMPYPNKPDQYINNEQKGFHLFGSMTNNGQATIEVEL 196 Query212 SKA 214  KA Sbjct 197 KKA 199(The above “Query” and “Sbjct” sequences disclosed as SEQ ID NOS 43-44,respectively.)4. Birds4a. Gallus gallus

>gi|118129726|ref|XP_001231839.1|PREDICTED: hypothetical protein isoform1 [Gallus gallus]

(SEQ ID NO: 45) MPPKEKKENDKPCNDNCQPKPQGKGVESLMKNIDVCRSVGLEIINRTRTVTLTDFRSYCFSGKIVTTLPFEIGPDSKGICIFAKTPYSLRGSVGTVVCKADTFFLAITFSNPYDYILYKIEFALEIFTEPNHLGNLGDVFSKMMKSKPYCGSSLFQRAVLESEHETLEVSKGSIRVQAKMSNNRKAIL KVQVEDMDPPPYSKGM

>ref|XP_001231839.1|UniGene infoGene info PREDICTED: hypotheticalprotein isoform 1 [Gallus gallus]

Length=204

GENE ID: 769729 LOC7697291 hypothetical protein LOC769729 [Gallusgallus]

Score=150 bits (378), Expect=9e-35, Method: Composition-based stats.

Identities=33/172 (19%), Positives=63/172 (36%), Gaps=22/172 (12%)

Query  58 LEALGNVKRKIAVGVDNES-GKTWTALNTYFRSGTSDIVLPHKVPHGKALLYNGQKDRGP116 L +V R + + + N + T T +Y SG LP ++ + K Sbjct  29LMKNIDVCRSVGLEIINRTRTVTLTDFRSYCFSGKIVTTLPFEIGPDSKGICIFAKTP-Y  87 Query117 VATGAVGVLAYLMSDGNTLAVLFSVPYDYNWYSNWWNVRIYKGKRRADQ-----RMYEEL 171 G+VG + +D LA+ FS PYDY Y + + 1+ + ++ ++ Sbjct  88SLRGSVGTVVCK-ADTFFLAITFSNPYDYILYKIEFALEIF---TEPNHLGNLGDVFSKM 143 Query172 YYNLSPFRG----------DNGWHTRNLGYGLKSRGFMNSSGHAILEIHVSK 213 P+ G ++ +M+++ AIL++ V Sbjct 144MK-SKPYCGSSLFQRAVLESEHETLEVSKGSIRVQAKMSNNRKAILKVQVED 194(The above “Query” and “Sbjct” sequences disclosed as SEQ ID NOS 37 and46, respectively.)5. Platypus5a. Ornithorhynchus anatinus

>gi|149491241|ref|XP_001516906.1|PREDICTED: hypothetical protein[Omithorhynchus anatinus]

(SEQ ID NO: 47) MAQTIEHLVHEVEAGRCVGIEITNTTNMTFRSPRTFCFSGHTLTPPTPIIHPNNAGFCIFVKRKFSLRGSVGLLVYEIEDQTLAIMFSNPFDYNFFKVEFAVALSGYKEETQDLKAFFELLYHEKQKGWLKMAKEKLCECQCPVSLENNGIRVTATMSNNAKAIIKLSSPDAKPPEGDVADVQPTTVRRPNPPPFPSPRPRIGSDLTGDQLATLDFESGK

>ref|XP_001516906.1|Gene info PREDICTED: hypothetical protein[Omithorhynchus anatinus]

Length=220

GENE ID: 100086848 LOC100086848|hypothetical protein LOC100086848[Ornithorhynchus anatinus]

Score=168 bits (426), Expect=2e-40, Method: Composition-based stats.

Identities=36/167 (21%), Positives=69/167 (41%), Gaps=12/167 (7%)

Query  58 LEALGNVKRKIAVGVDNESGKTWTALNTYFRSGTSDIVLPHKVPHGKALLYNGQKDRGPV117 L R + + + N + T+ + T+ SG + + A K R Sbjct   8LVHEVEAGRCVGIEITNTINMTFRSPRTFCFSGHTLTPPTPIIHPNNAGFCIFVK-RKFS  66 Query118 ATGAVGVLAYLMSDGNTLAVLFSVPYDYNWYSNWWNVRI--YKGKRRADQRMYEELYYNL 175 G+VG+L Y + D TLA++FS P+DYN++ + V + YK + + + +E L Y+ Sbjct  67LRGSVGLLVYEIED-QTLAIMFSNPFDYNFFKVEFAVALSGYKEETQDLKAFFELLYHEK 125 Query176 --------SPFRGDNGWHTRNLGYGLKSRGFMNSSGHAILEIHVSKA 214 + G++ M+++ AI+++A Sbjct 126 QKGWLKMAKEKLCECQCPVSLENNGIRVTATMSNNAKAIIKLSSPDA 172(The above “Query” and “Sbjct” sequences disclosed as SEQ ID NOS 48-49,respectively.)

As used herein, the term “isolated” refers to a composition that hasbeen removed from its in-vivo location (e.g. aquatic organism or moss).Preferably the isolated compositions of the present invention aresubstantially free from other substances (e.g., other proteins that donot comprise anti-adhesive effects) that are present in their in-vivolocation (i.e. purified or semi-purified).

As used herein the phrase “aquatic organism” refers to an organismliving in a water environment (marine or freshwater) such as for examplea fish or a sessile aquatic organism.

As used herein, the phrase “sessile aquatic organism” refers to anaquatic organism which is not freely moving for at least some a part ofits life cycle. Aquatic sessile organisms are usually permanentlyattached to a solid substrate of some kind, such as to a rock or thehull of a ship due to physical anchorage to the substrate, or for anyother reason (e.g. stone fish).

Exemplary sessile organisms include, but are not limited to, sessilecnidarians such as corals, sea anemones (e.g. Actinia equine andAiptasia pulchella), sea pens, aquatic sessile larva (e.g., jellyfishlarva), tube dwelling anemones and hydroids (e.g. Chlorohydraviridissima and Hydra vulgaris).

Exemplary fish that may be used according in embodiments of the presentinvention are preferably those dwelling in shallow waters or those thathide at the bottom layer of the ocean, sometimes in holes or caves. Suchfish include eel and catfish.

As used herein the phrase “moss” refers to a non-vascular plant of thebryophyta division, including any of the classes takakiposida,sphyagnopsida, andreaeopsida, anderaeobryopsida, polytirchopsida, orbryopsisa.

The moss may comprise, for example, Physcomitrella patens, Funariahygrometrica; Eukaryota; Viridiplantae; Streptophyta; Embryophyta;Bryophyta; Moss Superclass V; Bryopsida; Funariidae; Funariales;Funariaceae; or Physcomitrella.

The compositions of the present invention may also be expressed in-vivousing genetic engineering techniques (e.g. using transgenic aquaticsessile organisms).

According to some embodiments of the present invention, the compositionsof the present invention are devoid of cytotoxic or cytostatic activity,e.g. they are not bactericidal or bacteriostatic.

According to some embodiments of the present invention, the compositionsof the present invention are resistant to lyophilization—e.g. theiractivities are preserved following freeze drying.

As used herein the phrase “single cell organism” refers to a unicellularorganism also termed a microorganism or a microbe. The single cellorganism of the present invention can be a eukaryotic single cellorganism (e.g., protozoa or fungi for example yeast) or a prokaryoticsingle cell organism (e.g., bacteria or archaea). The single cellorganisms of the present invention may be in any cellular environment,such as for example, in a biofilm, as isolated cells or as a cellsuspension.

As used herein the term “biofilm” refers to an extracellular matrix Inwhich microorganisms are dispersed and/or form colonies. The biofilmtypically is made of polysaccharides and other macromolecules.

Exemplary bacterial cells, whose adhesion may be prevented according tothe method of the present invention, include gram positive bacteria andgram negative bacteria.

The term “Gram-positive bacteria” as used herein refers to bacteriacharacterized by having as part of their cell wall structurepeptidoglycan as well as polysaccharides and/or teichoic acids and arecharacterized by their blue-violet color reaction in the Gram-stainingprocedure. Representative Gram-positive bacteria include: Actinomycesspp., Bacillus anthracia, Bifidobacterium spp., Clostridium botulinum,Clostridium perfringens, Clostridium spp., Clostridium tetani,Corynebacterium diphtheriae, Corynebacterium jeikeium, Enterococcusfaecal is, Enterococcus faecium, Erysipelothrix rhusiopathiae,Eubacterium spp., Gardnerella vaginalis, Gemella morbillorum,Leuconostoc spp., Mycobacterium abscessus, Mycobacterium avium complex,Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacteriumhaemophilium, Mycobacterium kansasii, Mycobacterium leprae,Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacteriumsmegmatis, Mycobacterium terrae, Mycobacterium tuberculosis,Mycobacterium ulcerans, Nocardia spp., Peptococcus niger,Peptostreptococcus spp., Proprionibacterium spp., Sarcina lutea,Staphylococcus aureus, Staphylococcus auricularis, Staphylococcuscapitis, Staphylococcus cohnii, Staphylococcus epidermidis,Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcuslugdanensis, Staphylococcus saccharolyticus, Staphylococcussaprophyticus, Staphylococcus schleiferi, Staphylococcus similans,Staphylococcus warneri, Staphylococcus xylosus, Streptococcus agalactiae(group B Streptococcus), Streptococcus anginosus, Streptococcus bovis,Streptococcus canis, Streptococcus equi, Streptococcus milleri,Streptococcus mitior, Streptococcus mutans, Streptococcus pneumoniae,Streptococcus pyogenes (group A Streptococcus), Streptococcussalivarius, Streptococcus sanguis.

The term “Gram-negative bacteria” as used herein refer to bacteriacharacterized by the presence of a double membrane surrounding eachbacterial cell. Representative Gram-negative bacteria includeAcinetobacter calcoaceticus, Acinetobacter baumannii, Actinobacillusactinomycetemcomitans, Aeromonas hydrophila, Alcaligenes xylosoxidans,Bacteroides, Bacteroides fragilis, Bartonella bacilliformis, Bordetellaspp., Borrelia burgdorferi, Branhamella catarrhalis, Brucella spp.,Campylobacter spp., Chalmydia pneumoniae, Chlamydia psittaci, Chlamydiatrachomatis, Chromobacterium violaceum, Citrobacter spp., Eikenellacorrodens, Enterobacter aerogenes, Escherichia coli, Flavobacteriummeningosepticum, Fusobacterium spp., Haemophilus influenzae, Haemophilusspp., Helicobacter pylori, Klebsiella pneumoniae, Klebsiella spp.,Legionella spp., Leptospira spp., Moraxella catarrhalis, Morganellamorganii, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseriameningitidis, Pasteuretlaniultocida, Plesiomonas shigelloides,Prevotella spp., Proteus spp., Providencia rettgeri, Pseudomonasaeruginosa, Pseudomonas spp., Rickettsia prowazekii, Rickettsiarickettsii, Rochalimaea spp., Salmonella spp., Salmonella typhi,Serratia marcescens, Shigella spp., Shigella sonnei, Treponema carateum,Treponema pallidum, Treponema pallidum endemicum, Treponema pertenue,Veillonella spp., Vibrio cholerae, Vibrio vulnificus, Yersiniaenterocolitica, Yersinia pestis.

The term “fungi” as used herein refers to the heterotrophic organismscharacterized by the presence of a chitinous cell wall, and in themajority of species, filamentous growth as multicellular hyphae.Representative fungi whose adhesion may be prevented according to themethod of the present invention include Candida albicans, Saccharomycescerevisiae, Candida glabrata, Candida parapsilosis and Candidadubliniensis.

As used herein the phrase “preventing adhesion” refers to reducing oreliminating cell attachment to a surface (e.g. by reducing the rate ofgrowth on a surface). Preferably, the compositions of the presentinvention prevent cell adhesion by as much as 10%, more preferably by20%, more preferably by 30%, more preferably by 40%, more preferably by50%, more preferably by 60%, more preferably by 70%, more preferably by80%, more preferably by 90% and most preferably by 100% as measured by acell adhesion assay. Exemplary cell adhesion assays are described hereinbelow and in the Examples section that follows. It will be appreciatedthat the compositions of the present invention may also be capable ofpreventing cell aggregation (i.e. cell aggregation not to a surface).

The present invention contemplates prevention of cellular adhesion to awide variety of surfaces including fabrics, fibers, foams, films,concretes, masonries, glass, metals, plastics, polymers, and like.

According to one embodiment, the surface is comprised in a device thatis susceptible to biofilm formation. Exemplary devices whose surfacesare contemplated by the present invention include, but are not limitedto, vessel hulls, automobile surfaces, air plane surfaces, membranes,filters, and industrial equipment. The surface may also be comprised inmedical devices, instruments, and implants. Examples of such medicaldevices, instruments, and implants include any object that is capable ofbeing implanted temporarily or permanently into a mammalian organism,such as a human. Representative medical devices, instruments, andimplants that may be used according to the present invention include,for example, central venous catheters, urinary catheters, endotrachealtubes, mechanical heart valves, pacemakers, vascular grafts, stents andprosthetic joints. Methods of preventing cell attachment to medicaldevices and further examples thereof are described herein below.

According to another embodiment the surface is comprised in a biologicaltissue, such as for example, mammalian tissues e.g. the skin.

As mentioned, the method of the present invention is effected bycontacting the cell with a composition from an organism capable ofpreventing adhesion of the cell to a surface.

As used herein the term “contacting” refers to the positioning of thecompositions of the present invention such that they are in direct orindirect contact with the adhesive cells in such a way that the activeagent comprised within is able to prevent adhesion of cells thereto.Thus, the present invention contemplates both applying the compositionsof the present invention to a desirable surface and/or directly to theadhesive cells. The contacting may be effected in vivo (i.e. within amammalian body), ex vivo (i.e. in cells removed from the body) and/or invitro (i.e. outside a mammalian body).

Contacting the compositions with a surface can be effected using anymethod known in the art including spraying, spreading, wetting,immersing, dipping, painting, ultrasonic welding, welding, bonding oradhering. The compositions of the present invention may be attached asmonolayers or multiple layers.

According to one embodiment, the compositions of the present inventionmay be comprised in a whole living organism. For example, the presentinvention contemplates adding live aquatic organisms to an underwaterenvironment such that they are able to contact a surface and/or cellsadhered thereto (e.g. underwater pipes, ship hull) preventingmicroorganism adhesion thereto. It will be appreciated that the activeagent may be secreted from the aquatic organism. In this case, theaquatic organism does not have to be in direct contact with the surfaceor microorganism cells, but in sufficient proximity such that the activeagent is able to diffuse to its site of action. Thus, the compositionsof the present invention may be secreted into water and used in waterpurification treatments such as for example desalination of sea water orbrackish water.

According to a further aspect of the present invention, there isprovided a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or diluent and as an active ingredient a peptideisolated from an isolated natural peptide, said peptide comprising asequence selected from the group consisting of YDYNWY (SEQ ID NO: 1),YDYNLY (SEQ ID NO: 2), FDYNFY (SEQ ID NO: 3), FDYNLY (SEQ ID NO: 4),WDYNLY (SEQ ID NO: 8), FDYNWY (SEQ ID NO: 5), YDWNLY (SEQ ID NO: 6) andYDWHLY (SEQ ID NO: 7), or any other sequence as described herein.

According to other embodiments of the present invention, the abovepeptides may optionally be altered so as to form non-peptide analogs,including but not limited to replacing one or more bonds with lesslabile bonds, cyclization (described in greater detail below) and thelike. Additionally or alternatively, a peptide may optionally beconverted to a small molecule through computer modeling, as describedfor example in PCT Application No. WO/20071147098, hereby incorporatedby reference as if fully set forth herein.

A “peptidomimetic organic moiety” can optionally be substituted foramino acid residues in a peptide according to the present invention bothas conservative and as non-conservative substitutions. These moietiesare also termed “non-natural amino acids” and may optionally replaceamino acid residues, amino acids or act as spacer groups within thepeptides in lieu of deleted amino acids. The peptidomimetic organicmoieties optionally and preferably have steric, electronic orconfigurational properties similar to the replaced amino acid and suchpeptidomimetics are used to replace amino acids in the essentialpositions, and are considered conservative substitutions. However suchsimilarities are not necessarily required. The only restriction on theuse of peptidomimetics is that the composition at least substantiallyretains its physiological activity as compared to the native peptideaccording to the present invention.

Peptidomimetics may optionally be used to inhibit degradation of thepeptides by enzymatic or other degradative processes. Thepeptidomimetics can optionally and preferably be produced by organicsynthetic techniques. Non-limiting examples of suitable peptidomimeticsinclude D amino acids of the corresponding L amino acids, tetrazol(Zabrocki et al., J. Am. Chem. Soc. 110:5875 5880 (1988)); isosteres ofamide bonds (Jones et al., Tetrahedron Lett. 29: 3853 3856 (1988)); L L3 amino 2 propenidone 6 carboxylic acid (L L Acp) (Kemp et al., J. Org.Chem. 50:5834 5838 (1985)). Similar analogs are shown in Kemp et al.,Tetrahedron Lett. 29:5081 5082 (1988) as well as Kemp et al.,Tetrahedron Lett. 29:5057 5060 (1988), Kemp et al., Tetrahedron Lett.29:4935 4938 (1988) and Kemp et al., J. Org. Chem. 54: 109 115 (1987).Other suitable but exemplary peptidomimetics are shown in Nagai andSato, Tetrahedron Lett. 26:647650 (1985); Di Maio et al., J. Chem. Soc.Perkin Trans., 1687 (1985); Kahn et al., Tetrahedron Lett. 30:2317(1989); Olson et al., J. Am. Chem. Soc. 112:323 333 (1990); Garvey etal., J. Org. Chem. 56:436 (1990). Further suitable exemplarypeptidomimetics include hydroxy 1,2,3,4 tetrahydroisoquinoline 3carboxylate (Miyake et al., J. Takeda Res. Labs 43:53 76 (1989));1,2,3,4 tetrahydro-isoquinoline 3 carboxylate (Kazmierski et al., J. Am.Chem. Soc. 133:2275 2283 (1991)); histidine isoquinolone carboxylic acid(HIC) (Zechel et al., Int. J. Pep. Protein Res. 43 (1991)); (2S, 3S)methyl phenylalanine, (2S, 3R) methyl phenylalanine, (2R, 3S) methylphenylalanine and (2R, 3R) methyl phenylalanine (Kazmierski and Hruby,Tetrahedron Lett. (1991)).

Exemplary, illustrative but non-limiting non-natural ammo acids includebeta-amino acids (beta3 and beta2), homo-amino acids, cyclic aminoacids, aromatic amino acids, Pro and Pyr derivatives, 3-substitutedAlanine derivatives, Glycine derivatives, ring-substituted Phe and TyrDerivatives, linear core amino acids or diamino acids. They areavailable from a variety of suppliers, such as Sigma-Aldrich (USA) forexample.

In the present invention any part of a peptide may optionally bechemically modified, i.e. changed by addition of functional groups. Themodification may optionally be performed during synthesis of themolecule if a chemical synthetic process is followed, for example byadding a chemically modified amino acid. However, chemical modificationof an amino acid when it is already present in the molecule (“in situ”modification) is also possible.

The amino acid of any of the sequence regions of the molecule canoptionally be modified according to anyone of the following exemplarytypes of modification (in the peptide conceptually viewed as “chemicallymodified”). Non-limiting exemplary types of modification includecarboxymethylation, acylation, phosphorylation, glycosylation or fattyacylation. Ether bonds can optionally be used to join the serine orthreonine hydroxyl to the hydroxyl of a sugar. Amide bonds canoptionally be used to join the glutamate or aspartate carboxyl groups toan amino group on a sugar (Garg and Jeanloz, Advances in CarbohydrateChemistry and Biochemistry, Vol. 43, Academic Press (1985); Kunz, Ang.Chem. Int. Ed. English 26:294-308 (1987)). Acetal and ketal bonds canalso optionally be formed between amino acids and carbohydrates. Fattyacid acyl derivatives can optionally be made, for example, by acylationof a free amino group (e.g., lysine) (Toth et al., Peptides: Chemistry,Structure and Biology, Rivier and Marshal, eds., ESCOM Publ., Leiden,1078-1079 (1990)).

As used herein the term “chemical modification,” when referring to apeptide according to the present invention, refers to a peptide where atleast one of its amino acid residues is modified either by naturalprocesses, such as processing or other post-translational modifications,or by chemical modification techniques which are well known in the art.Examples of the numerous known modifications typically include, but arenot limited to: acetylation, acylation, amidation, ADP-ribosylation,glycosylation, GPI anchor formation, covalent attachment of a lipid orlipid derivative, methylation, myristylation, pegylation, prenylation,phosphorylation, ubiquitination, or any similar process.

According to some embodiments of this aspect of the present invention,there is provided a method of preventing or treating a pathogeninfection in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of thepharmaceutical composition, thereby treating or preventing the pathogeninfection.

According to alternative embodiments of this aspect of the presentinvention, there is provided a method of preventing attachment ofexogenous bacteria to the gastrointestinal tract.

The mammalian gastrointestinal tract contains a wide variety ofindigenous microflora, which provide resistance to colonization byenteric pathogen. In return for providing the host with enhanced defenseagainst pathogens, the indigenous microflora gain access to anutrient-enriched, stable environment, and thereby enter a symbioticrelation with the host's intestinal tract.

Symbiotic bacteria attach to the gastrointestinal epithelium in humansby high-affinity, receptor-mediated attachment. In contrast, exogenousbacteria attach to the epithelium by a low-affinity mechanism. Withoutwishing to be limited by a single hypothesis, the compositions of thepresent invention are expected to selectively prevent or decrease thislow-affinity attachment, thereby preventing the initial step of biofilmformation.

The composition of the present invention is therefore useful fortreatment or prevention of diseases of the gastrointestinal tract, suchas, for example, Crohn's disease or ulcerative colitis, including, forexample, collagneous colitis, lymphocytic colitis, ischaemic colitis,diversion colitis, infective colitis and Behcet's syndrome.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

As used herein the term “active ingredient” refers to the organismcompositions (and agents purified therefrom) accountable for theintended biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier,” which may be usedinterchangeably, refer to a carrier or a diluent that does not causesignificant irritation to an organism and does not abrogate thebiological activity and properties of the administered compound. Anadjuvant is included under these phrases.

Herein, the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils, and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found Inthe latest edition of “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., which is herein fully incorporated byreference and are further described herein below.

As mentioned, the pharmaceutical compositions of the present inventionmay be administered to a subject in need thereof in order to prevent ortreat a pathogen infection.

As used herein the term “subject in need thereof’ refers to a mammal,preferably a human subject.

As used herein the term “treating” refers to curing, reversing,attenuating, alleviating, minimizing, suppressing or halting thedeleterious effects of a pathogen infection.

As used herein the phrase “pathogen infection” refers to any medicalcondition which is caused by a pathogenic organism. Examples of pathogeninfections include, but are not limited to, chronic infectious diseases,subacute infectious diseases, acute infectious diseases, viral diseases,bacterial diseases, protozoan diseases, parasitic diseases, fungaldiseases, mycoplasma diseases, archaea diseases and prion diseases.

According to one embodiment, the pathogen infection is caused by anorganism capable of growing in or on a biofilm.

Examples of pathogen infections caused by microbial biofilms includenative valve endocarditis (NVE), otitis media (OM), chronic bacterialprostatitis, cystic fibrosis (CF) and periodontitis. Additional pathogeninfections that are not specifically attributed to biofilms include, butare not limited to urinary infections, female genital tract infectionsand pneumonia. Infections due to implantation of medical devices includevascular catheter infections, arterial prosthetic infections, infectionsof prosthetic heart valves, prosthetic joint infections, infections ofcentral nervous system shunts, orthopedic implant infections, pacemakerand defibrillator infections, hemodialysis and peritoneal dialysisinfections, ocular infections, urinary tract infections, infections ofthe female genital tract, infections associated with endotrachealintubation and tracheostomy and dental infections.

As used herein the phrase “pathogenic organism” refers to any singlecell organism which is capable of causing disease, especially a livingmicroorganism such as a bacteria or fungi. Preferably the pathogenicorganism is capable of growing in or on a biofilm. Many commonpathogenic organisms exist in mammals (e.g. humans) as biofilms andcause disease. These include, but are not limited to, Mannheimiahaemolytica and Pasteurella multocida (causing pneumonia), Fusobacteriumnecrophorum (causing liver abscess), Staphylococcus aureus andPseudomonas aeruginosa (causing wound infections), Escherichia coli andSalmonella spp (causing enteritis), Staphylococcus aureus andStaphylococcus epidermidis (causing OM), and Streptococci sp.,Staphylococci sp., Candida, and Aspergillus sp. (causing NVE).

It will be appreciated that treatment of infectious diseases accordingto the present invention may be combined with other treatment methodsknown in the art (i.e., combination therapy). These include, but are notlimited to, antimicrobial agents such as penicillins, cephalosporins,carbapenems, aminoglycosides, macrolides, lincomycins, tetracyclines,chloramphenicol, and griseofulvin.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal, or parenteraldelivery, including intramuscular, subcutaneous, and intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, inrtaperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations that can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For topical administration, the compositions of the present inventionmay be formulated as a gel, a cream, a wash, a rinse or a spray.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries as desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, and sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents, such ascross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof, such as sodium alginate, may be added.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate, and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane, or carbon dioxide. In the case of apressurized aerosol, the dosage may be determined by providing a valveto deliver a metered amount. Capsules and cartridges of, for example,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base, such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with, optionally, anadded preservative. The compositions may be suspensions, solutions, oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing, and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water-based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters such as ethyl oleate, triglycerides, orliposomes. Aqueous injection suspensions may contain substances thatincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents that increase the solubility ofthe active ingredients, to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., a sterile, pyrogen-free,water-based solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, for example, conventional suppository bases such as cocoabutter or other glycerides.

Pharmaceutical compositions suitable for use in the context of thepresent invention include compositions wherein the active ingredientsare contained in an amount effective to achieve the intended purpose.More specifically, a “therapeutically effective amount” means an amountof active ingredients (e.g., an aquatic organism composition or a mosscomposition) effective to prevent, alleviate, or ameliorate symptoms ofa pathogenic infection (e.g., fever) or prolong the survival of thesubject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, the dosage orthe therapeutically effective amount can be estimated initially from invitro and cell culture assays. For example, a dose can be formulated inanimal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration, and dosage canbe chosen by the individual physician in view of the patient'scondition. (See, e.g., Fingl, E. et al. (1975), “The PharmacologicalBasis of Therapeutics,” Ch. 1, p.1).

Dosage amount and administration intervals may be adjusted individual toprovide sufficient plasma or brain levels of the active ingredient toinduce or suppress the biological effect (i.e., minimally effectiveconcentration, MEC). The MEC will vary for each preparation, but can beestimated from in vitro data. Dosages necessary to achieve the MEC willdepend on individual characteristics and route of administration.Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks, oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA-approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser device may also be accompaniedby a notice in a form prescribed by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions for human orveterinary administration. Such notice, for example, may includelabeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert. Compositionscomprising a preparation of the invention formulated in apharmaceutically acceptable carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition, as further detailed above.

As mentioned, medical devices and implants are commonly infected withopportunistic bacteria and other infectious microorganisms (e.g., fungi)in some cases necessitating the removal of implantable devices. Suchinfections can also result in illness, long hospital stays, or evendeath. The prevention of biofilm formation and infection of medicaldevices is therefore highly desirous.

Thus, the present invention also contemplates medical devices in whichthe above-described compositions are attached thereto.

As used herein the term “medical device” refers to any implant,instrument, apparatus, implement, machine, device or any other similaror related object (including any component or accessory), which isintended for use in the diagnosis, treatment, cure or prevention ofdisease or other conditions. Such medical device is intended for use inman or other animals and is anticipated to affect the structure or anyfunction of the body. Such medical device does not achieve its primaryintended purposes through chemical action and is not dependent uponbeing metabolized for the achievement of its primary intended purposes.

As used herein the term “implant” refers to any object intended forplacement in a human body that is not a living tissue. The implant maybe temporary or permanent. An implant can be an article comprisingartificial components, such as catheters or pacemakers. Implants canalso include naturally derived objects that have been processed so thattheir living tissues have been devitalized. As an example, bone graftsthat have been processed so that their living cells are removed(acellularized), but so that their shape is retained to serve as atemplate for ingrowth of bone from a host. As another example, naturallyoccurring coral can be processed to yield hydroxyapatite preparationsthat can be applied to the body for certain orthopedic and dentaltherapies.

The present invention therefore envisions coating medical devices withthe compositions of the present invention to prevent cell adherencethereto so as to reduce/eliminate any possible cell aggregation andbiofilm formation known to occur following implantation. Device-relatedinfections usually result from the introduction of microorganisms,primarily bacteria, during the device insertion or implantationprocedure, or from attachment of blood-borne organisms to the newlyinserted device and their subsequent propagation on its surface. Coatingthe medical device with the compositions of the present invention willtherefore inhibit biofilm formation of one or more microbial species,will prevent medical device related infections, and consequently willreduce the need of antibiotic treatment or removal of the medical devicefrom the subject.

Medical devices that may be coated according to the teachings of thepresent invention include, but not limiting to, artificial bloodvessels, catheters and other devices for the removal or delivery offluids to patients, artificial hearts, artificial kidneys, orthopedicpins, prosthetic joints, plates and implants; catheters and other tubes(including urological and biliary tubes, endotracheal tubes,peripherably insertable central venous catheters, dialysis catheters,long term tunneled central venous catheters, peripheral venouscatheters, short term central venous catheters, arterial catheters,pulmonary catheters, Swan-Ganz catheters, urinary catheters, peritonealcatheters), urinary devices (including long term urinary devices, tissuebonding urinary devices, artificial urinary sphincters, urinarydilators), shunts (including ventricular or arterio-venous shunts);prostheses (including breast implants, penile prostheses, vasculargrafting prostheses, aneurysm repair devices, mechanical heart valves,artificial joints, artificial larynxes, otological implants),anastomotic devices, vascular catheter ports vascular stents, clamps,embolic devices, wound drain tubes, ocular lenses, dental implants,hydrocephalus shunts, pacemakers and implantable defibrillators,needleless connectors, voice prostheses and the like.

Another possible application of the compositions of the presentinvention is the coating of surfaces found in the medical and dentalenvironment. Such surfaces include the inner and outer aspects ofvarious instruments and devices, whether disposable or intended forrepeated uses. Such surfaces include the entire spectrum of articlesadapted for medical use, including without limitation, scalpels,needles, scissors and other devices used in invasive surgical,therapeutic or diagnostic procedures; blood filters. Other examples willbe readily apparent to practitioners in these arts.

Surfaces found in the medical environment also include the inner andouter aspects of pieces of medical equipment, medical gear worn orcarried by personnel in the health care setting. Such surfaces caninclude surfaces intended as biological barriers to infectious organismsin medical settings, such as gloves, aprons and faceshields. Commonlyused materials for biological barriers are thermoplastic or polymericmaterials such as polyethylene, dacron, nylon, polyesters,polytetrafluoroethylene, polyurethane, latex, silicone and vinyl. Othersurfaces can include counter tops and fixtures in areas used for medicalprocedures or for preparing medical apparatus, tubes and canisters usedin respiratory treatments, including the administration of oxygen, ofsolubilized drugs in nebulizers and of anesthetic agents. Other suchsurfaces can include handles and cables for medical or dental equipmentnot intended to be sterile. Additionally, such surfaces can includethose non-sterile external surfaces of tubes and other apparatus foundin areas where blood or body fluids or other hazardous biomaterials arecommonly encountered.

The compositions of the present invention can be used on the surface ofor within these medical devices to provide long term protection againstmicroorganism colonization and reduce the incidence of device-relatedinfections. These compositions can also be incorporated in combinationwith an anti-microbial agent (e.g., antibiotic agent) into coatings formedical devices. Such a combination will sufficiently kill or inhibitthe initial colonizing bacteria and prevent device-related infections aslong as the substance is presented in an inhibitory concentration at thedevice-microbe interface.

The compositions of the present invention can be directly incorporatedinto the polymeric matrix of the medical device at the polymer synthesisstage or at the device manufacture stage. The compositions can also becovalently attached to the medical device polymer. These and many othermethods of coating medical devices are evident to one of ordinary skillin the ant.

Additional surfaces that can be treated according to the teachings ofthe present invention include the inner and outer aspects of thosearticles involved in water purification, water storage and waterdelivery, and those articles involved in food processing. Thus thepresent invention envisions coating a solid surface of a food orbeverage container to extend the shelf life of its contents.

Surfaces related to health can also include the inner and outer aspectsof those household articles involved in providing for nutrition,sanitation or disease prevention. Thus, the compositions of the presentinvention can be used for removal of disease-causing microorganisms fromexternal surfaces. These can include, for example food processingequipment for home use, materials for infant care, tampons, soap,detergents, health and skincare products, household cleaners and toiletbowls.

The surface may be also be laboratory articles including, but notlimited to, microscopic slide, a culturing hood, a Petri dish or anyother suitable type of tissue culture vessel or container known in theart.

The inventors of this application also envision the use of thecompositions of the present invention as anti-fouling agents.

As used herein the term “anti-fouling agents” refers to the compoundsused to protect underwater surfaces from attaching single cellorganisms. These single cell organisms include microorganism such asbacteria and fungi.

These underwater surfaces include any water immersed surface, includingships'boats's hulls (i.e., the body or frame of a ship or boat),submergence vehicles, navigational aids, screens, nets, constructions,floating or emplaced offshore platforms (e.g., docks), buoys, signalingequipment and articles which come into contact with sea water or saltywater. Other underwater surfaces include structures exposed to sea waterincluding pilings, marine markers, undersea conveyances like cabling andpipes, fishing nets, bulkheads, cooling towers, and any device orstructure that operates submerged.

The compositions of the present invention can be incorporated intomarine coatings to limit undesirable marine fouling. Thus, theanti-fouling agents of the present invention can be formulated so as notto contain toxic materials (such as heavy metals), and still retaintheir efficacy. The anti-fouling paint of the present invention mayfurther contain binders(s), pigment(s), solvent(s) and additive(s).

Examples of solvents that may be used include aromatic hydrocarbons suchas xylene and toluene; aliphatic hydrocarbons such as hexane andheptane, esters such as ethyl acetate and butyl acetate; amides such asN-methylpyrrolidone and N,N-dimethylformamide; alcohols such asisopropyl alcohol and butyl alcohol; ethers such as dioxane, THF anddiethyl ether; and ketones such as methyl ethyl ketone, methyl isobutylketone and methyl isoamyl ketone. The solvents may be used alone or incombination thereof.

Examples of binders that may be used include alkyd resin, acrylic orvinyl emulsions, polyurethane resins, epoxy resins, silicone basedresins, acrylic resins, inorganic silicate based resins, vinyl resins,particularly a vinyl chloride/vinyl acetate copolymer, and rosin.

Examples of pigments that may be used include titanium dioxide, cuprousoxide, iron oxide, talc, aluminum flakes, mica flakes, ferric oxide,cuprous thiocyanate, zinc oxide, cupric acetate meta-arsenate, zincchromate, zinc dimethyl dithiocarbamate, zinc ethylenebis(dithiocarbamate) and zinc diethyl dithiocarbamate.

Examples of additives that may be incorporated into the coatingcomposition include dehumidifiers, wetting/dispersing agents,anti-settling agents, anti-skinning agents, drying/curing agents,anti-marring agents and additives ordinarily employed in coatingcompositions as stabilizers and anti-foaming agents. Additionally, anyantibiotic which is relatively insoluble in seawater can be used with ananti-fouling marine paint.

Methods of preparing marine anti-fouling paints are explained in detailin U.S. Pat. Nos. 4,678,512; 4,286,988; 4,675,051; 4,865,909; and5,143,545.

The compositions of the present invention may also be used for providingantibacterial properties in cosmetics, to prevent spoiling of theproduct.

The compositions may further be used to provide an antibacterial effectto the mouth, teeth and gums, such as by incorporation in a toothpaste,mouthwash, or chewing gum. Taken together the present teachings portraya wide range of novel anti-adhesive agents isolated from organisms suchas aquatic organisms and moss. The broad spectrum of the anti adhesioneffects of these agents (e.g. inhibiting adhesion of gram positive andgram negative bacteria) together with their ability to effect theinitial, vulnerable stages of microbial biofilm formation, makes theseagents prime candidates as anti-biofilm agents. Moreover, theanti-adhesive agents described herein are clonable enablingmodifications and mass production thereof. In addition their stability(i.e. resistance to environmental conditions) makes these agentssuitable for a diverse array of applications.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology,” JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning,” John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA,” Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series,” Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook,” Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes Coligan J.E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology”(8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell andShiigi (eds), “Selected Methods in Cellular Immunology,” W. H. Freemanand Co., New York (1980); available immunoassays are extensivelydescribed in the patent and scientific literature, see, for example,U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987;3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1317,Academic Press; “PCR Protocols: A Guide To Methods And Applications,”Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

EXAMPLES

Reference is now made to the following examples, which together with theabove description, illustrate the invention in a non limiting fashion.

Example 1 MS/MS Analysis of an Active Fraction Extracted from AiptesiaAnemone

Crude extract of Aiptasia pulchella (whole organism) was separated onSephadex G-10 column resulted in 2 fractions, both exhibitinganti-adherence/biofilm formation activity (FIG. 13).

Rechromatography of the high molecular fraction from Sephadex G-10 onSephadex G-75 resulted in two main peaks representing high and lowmolecular fractions (FIG. 14).

Reversed phase high performance liquid chromatography (RP-HPLC)separation with c-18 column, of the low molecular fraction from the G-75column, in linear gradients of acetonitrile (3-80% from 5 to 75 minutes)in 0.1% TFA at a flow rate of 2 ml/min, resulted in several activefractions as anti adhesive compounds on Pseudomonas aeruginosa ATCC27853. Fractions were collected every 2 minutes (FIG. 15).

All active fractions where digested by trypsin, analyzed by LC-MS/MS onQtof Premier (Waters) and on LTQ-Orbitrap (Thermo) and identified byPep-Miner and Sequest software against the Eukaryotes part of the nrdatabase. Active fraction eluted at 72.3% acetonitrile (marked in redarrow) was found to be similar to Equinatoxin 5 from Actinia equine.

Example 2 Identification of a Conserved Region of Anemone Cytotoxin

Purified template DNA was prepared from 25 mg of Aiptasia pulchella andAnemonia viridans using the wizard genomic DNA purification kit(Promega, USDA), according to the manufacturer's protocol for isolationof genomic DNA from animal tissue. PCR was carried out on 500 ng ofpurified template DNA from Aiptasia pulchella and Anemonia viridansusing Reddy Mix PCR master mix (ABgene, UK), with the followingprotocol: 95° C.-5 min (95° C. 30 sec, 52° C. 30 sec and 72° C. lmin)X35, 72° C. for 10 min.

Primers Eqt-F (GTR TCG ACA ACG AGT CRG G) (SEQ ID NO: 22) and Eqt-R252(TGA CAT YCC ACC AGT TGC TG) (SEQ ID NO: 23) were added to the reactionmixture, to a final concentration of 0.51 μM each.

Positive PCR reactions which gave DNA amplicon of size of ˜250 bp weresent for DNA sequencing.

A PCR amplicon from Aiptasia pulchella gave the following 265 bpsequence:

(SEQ ID NO: 50) GTGTCGCCAACGAGTCGGGATGCACTTGGGAAAAGCCAAATACATACTTCTTCTCTGGTACTGAGGTATAAAGTGCCTCCCTCTAAAGCTTGAGAATAAAAAAGCACTTTTGTACGGCCCACGTAAGACAACAGGGCCTGTTGCCACGGGAGCTGTTGGAGTGCTCACTTACAAAATGTTGTGCACCAATGAGACGAACACTCTGGCTGTTCTTTTCAGTGTACCCTTCGACTACAACTTGTACAGCAACTGGTGGAAATGTCAA

BLASTx comparison of the predicted amino acid sequence encoded by theabove polynucleotide sequence to known protein sequences in the GeneBankprovided the following results: Identities=54/88 (61%), Positives=62/88(70%). To other anemone cytotoxins like: hemolytic toxin [Actineriavillosa], PsTX-20A [Phyllodiscus semoni], cytolysin I precursor[Sagartia rosea] and equinatoxin IV precursor [Actinia equina];(accession numbers: BAD74019.1, BAC45007.1, AAP04347.1 and AF057028_1).

The relevant peptide sequence [FSVPFDYNLYSNWW] (SEQ ID NO: 51) appears mthe Aiptasial sequence.

PCR amplicon from Anemonia viridans gave the following 254 bp sequence:

(SEQ ID NO: 52) TGTGTCGACAACGAGTCgGGCaagacgtGgaCCGCAntgaaCACATACTTCCGTTCTGGcACCTCTGATnTCrTCCTTCCCCATACAGTTCCACATGGTAAGGCACTGCTCTACAACGGTCAGAAAGATCGTGGTCCAGTTGCGACTGGCGtgGTTGGAGTACTTGCTTATGcCATGAGCgATGGAAACACCCtGGCCGTTTTgTTCAGCrTTCCCTaTGACTATAACCtGTACA GCAACTGGTGGAATGTCAA.

BLASTn comparison to known nucleotide sequences in the GeneBank gavesimilarities of 97%, 96% and 95% to Equinatoxins 5 [accession number:AEU51900], 4 [accession number: AF057028] and 2 [accession number:AEU41661], in correspondence.

Predicted amino acid sequence based on translation of the secondpositive ORF gave the following AA sequence:CRQRVGMHLGKAKYILLLWY*GIKCLPLKLENKKALLYGPRKTTGPVATGAVGVLT YKMLCTNETNTLAVLFSVPFDYNLYSNWWKCQ (SEQ ID NOS 53 and 62, respectively, inorder of appearance).

Example 3 Comparison of Activity of Synthetic Peptides

Peptides listed below were synthesized using solid-phase methods andpurification to 90% scale was performed by Peptron Inc. (Taej eon,Korea).

The peptides were dissolved using 20 μl dimethyl sulfoxide (DMSO) anddiluted in double distilled-water to a concentration of 5 mg/ml. Furtherdilutions were performed in phosphate buffered saline (PBS).

The activities of the following synthetic peptides were studied on aclinical isolate of Acinetobacter Baumannii and Pseudomonas aeruginosaATCC 27853 at peptide concentrations ranging from 500-0.5 μg/ml.Peptides diluted to appropriate concentrations were incubated with thebacteria for 24-48 hours.

For bacterial adherence bioassys, biofilms were grown in 96-wellround-bottom polystyrene plates. Briefly, 180 μl of overnight cultureswere added to wells supplemented with 20 μl of appropriate peptidediluted in PBS. After 24 h of incubation at 37° C., each well was washedwith water and was stained with 250 μl of crystal violet solution. Thedye was then removed by thorough washing with water. For quantificationof attached cells, crystal violet was solubilized in 250 μl of 1% sodiumdodecyl sulfate (SDS) and the absorbance was measured at 595 nm.

AbacZ-17C (SEQ ID NO: 32) CMFSVPFDYNWYSNWWC AbacZ-15 (SEQ ID NO: 54)Ac-MFSVPFDYNWYSNWW-NH2 AbacZ-16C (SEQ ID NO: 55) CFSVPFDYNWYSNWWCAbacZ-6 (SEQ ID NO: 5) FDYNWY AbacZ-8C (SEQ ID NO: 56) CFDYNWYC

Results are shown in FIGS. 1 to 12. As seen in FIGS. 5 and 7, thepeptides did not kill or inhibit growth of bacteria. FIGS. 6, 8 and 10to 12, demonstrate that the peptides prevented the formation ofbiofilms.

Example 4 Identification of Preferred Peptides

In order to identify the most active cyclic peptides according to thepresent invention, a manual Parallel Peptide synthesizer with peptidepurifier is used to produce peptides which differ from each other inlength and cyclization strategy. Each peptide is screened for anti-30adhesive activity in microplate and flow cell assays, and selection ofhighly active peptides is performed. Computerized modeling of severalversions of peptide is used to optimize selection of the activecompounds.

For more sensitive screening the bioassay is scaled up using theBacTiter-Glo microbial cell viability Assay of Promega, USA. This methoduses more sensitive spectrometric technique based on luminescence.

Various cyclization strategies are used to obtain optimized cyclicpeptide from linear analog with satisfactory bioactivity. FIG. 16A showsthe generalized structure of the cyclic lead with emulsifier arm. Thelinear analog is a 14mer peptide with hydrophobic core (baa) that isdefined as a pharmaeophore. Cyclic leads are prepared, preserving thispharmacophore and an emulsifying arm (hydrophobic moiety) such aspolyethylene, polypropylene, Teflon etc. is then added to provideabsorbing capabilities to a hydrophobic polymeric surface (FIG. 16B).

Epitope mapping, escanning and Cycloscan methodologies are used forrevealing shorter, more cost-effective, peptides possessing the desiredbiological activity.

The cyclic lead is prepared using 9H-fluoren-9-ylmethoxycarbonyl solidphase peptide synthesis (Fmoc SPPS).

According to a representative procedure, as shown in FIG. 17, the firstprotected amino acid is condensed with chlorotitryl (Cl-Trt) resin usingN,N-diisopropylethylamine (DIEA) in dichloromethane (DCM) or with RinkAmide usingO-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate(HBTU) as coupling reagent.

The next couplings are performed using a standard Fmoc protocol with2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate Methanaminium (HATU) orbenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBoP), DIEA in N-methyl-2-pyrrolidinone (NMP). The allyloxycarbonyl(alloc) groups are deprotected by Pd-triphenylphosphine (Tetrakis) usingacetyl hydroxide/N-methyl maleimide/diethene chloride (AcOH/NMM/DCE)cocktail.

The cyclization step is carried out as a standard coupling reaction (inthe case of formation of an amide bond) or by bubbling oxygen (in thecase of formation of a disulfide bridge). Other types of cyclizations asknown in the art may also be performed.

The peptides are cleaved from the resin by treatment withtrifluoroacetic acid:dichloromethane:tri-iso-propylsilane (TFA:DCM:TIS),1:98:1 in the presence of 1,2-ethanedithiol for 30 min in case of theCl-Trt resin, and in the presence of 95% TFA, TIS and H₂O in the case ofRink Amide.

The crude product in the solution (AcOH/H₂O 1:1) is purified bypreparative HPLC or MPLC to afford pure cyclic peptide. The purity isdetermined by analytical HPLC. The structures are confirmed by LC-MS andAA analysis.

The next stage involves linkage of the hydrophobic arm for introducingabsorbing properties to the cyclic peptide lead. This arm is linkedusing standard SPPS protocol.

FIG. 17 shows a flowchart outlining the process for development of acyclic peptide lead with an emulsifying arm.

Example 5 Treatment of Water or Fluid Medium

The above peptides and/or compositions and/or organisms may optionallyand preferably be used to treat water and/or a fluid medium, or a systemor apparatus containing such, including but not limited to a reverseosmosis filter and/or filtration apparatus or system.

The effect of Actiniaria extracts on biofilm formation using polyamidecoupons in a flow cell without filtration is tested as follows. Theeffect of biofilm growth is analyzed in a flowcell dedicated forconfocal microscopy on a similar polyamide surface as an RO (reverseosmosis) active layer. A dual channel flowcell (FC 270, BiosurfaceTechnologies, Montana, USA) is operated with both model strains and realmicrobial inoculum taken from reverse osmosis (RO) membrane couponlocated in selected places on the Mediterranean sea supplemented withdifferent concentrations (from nanograms to micrograms per ml) of theextracts. The flow regime in the flowcells is laminar and similar to atypical RO operational flow conditions. For the model strains, seawatersynthetic media is determined (see (Fritzmann et al., 2007: Fritzmann,C., Lowenberg, J., Wintgens, T., and Melin, T. (2007) State-of-the-artof reverse osmosis desalination. Desalination 216: 1-76) and IDE reportside-tech.tech.com/) and are used for cell attachment biofilm growthexperiments. For the microbial consortium being isolated from thedesalination plant in Palmachim, real seawater is used as a media formicrobial attachment and biofilm growth experiments. Model strains to beused are Vibrio fisheri and Caulobacter crescentus. In the dual channelflowcell, one channel is supplemented with an extract and the otherserves as a control with only the solvent being added to the media (forexample, when the extract is dissolved in ethanol).

The flowcell biofilms are microscopically analyzed at different timepoints (up to 14 days of experiment) when viable cells, dead cells andExtra cellular polymeric substances (EPS) are stained with fluorescentprobes (different fluorescent labeled lectins are used for probingdifferent polysaccharide constitutents in the EPS) and visualized withlaser scanning confocal microscopy (LSCM). Microscopic analysis isperformed using image processing analysis softwares such as Imarisbitplane and COMSTAT (Heydorn et al., 2002: Ersboll, B., Kato, J.,Hentzer, M., Parsek, M. R., Tolker-Nielsen, T. et al. (2002) Statisticalanalysis of Pseudomonas aeruginosa biofilm development: impact ofmutations in genes involved in twitching motility, cell-to-cellsignaling, and stationary-phase sigma factor expression. Applied andEnvironmental Microbiology 68: 2008-2017).

Calcium, which has significant effect on the adhesiveness and thecompactness of the biofilm is also monitored and visualized with LSCMusing Calcium specific fluorochromes such as Fura-2 (Grynkiewicz et al.,1985: Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) A newgeneration of Ca2+ indicators with greatly improved fluorescenceproperties, Journal of Biological Chemistry 260: 3440-3450; Neu et al.,2002: Neu, T. R., Kuhlicke, U., and Lawrence, J. R. (2002) Assessment ofFluorochromes for Two-Photon Laser Scanning Microscopy of Biofilms,Applied and Environmental Microbiology 68: 901-909).

Antifouling properties are also examined by analyzing the adherence ofdifferent types of EPC/bacteria with QCM-D surface-modified crystalscovalently bonded to different peptides.

QCM-D employs an ultra-sensitive mass sensor (silica-coated quartzcrystal) housed inside a flow cell with a well-defined geometry andhydrodynamic characteristics, a design that allows real-time monitoringof mass adsorption with no required labeling. The piezoelectric quartzcrystal oscillates laterally with an amplitude of 1-2 nm when a voltageis applied to the electrodes affixed to the quartz crystal. Asdeposition (adsorption) occurs on the crystal surface, it leads to ashift in the vibrational frequency of the crystal. In addition tomonitoring the frequency shift to determine the adsorbed mass, thicknessand structural conformation of the adsorbed layer can be extracted bysimultaneous monitoring of the energy of dissipation, which is the sumof all energy losses within the system per oscillation cycle.Intriguingly, in addition to measuring the adsorption and adherence ofthe different types of EPS (i.e., changes in polysaccharides/proteincontents) under different environmental conditions (i.e., changes indivalent cation concentrations), QCM-D can also reveal the visco-elasticproperties, conformational changes, and thickness of the precipitatednano-layer.

Example 6 Effect of Active Peptides on Reverse Osmosis Biofouling UnderDesalination Conditions

The effect of Actiniaria extracts on reverse osmosis biofouling underdesalination conditions is determined.

Two RO (reverse osmosis) bench-scale units are operated for desalinationof seawater, and biofouling experiments with both candidate modelstrains and microbial consortium isolated from the GES desalinationplant (as mentioned above) are conducted both in a synthetic seawatermedia and a real seawater. Commercialized flat-sheet membranes SW-30 ofDow-Filmtec are used for these biofouling experiments. Specific measuresof process conditions are obtained: permeate flux, total organic carbon(TOC), oxygen concentrations in the permeate and in the brine solution,oxygen uptake rate, and the rejection of different ions and cations bythe membrane. Different biofilm components are analyzed: Chemicalanalysis of the biofouling layer will include characterization ofproteins, carbohydrates, lipids, and DNA. Microscopic observation andanalysis is performed as mentioned above.

While the invention has been described with respect to a limited numberof embodiments, it will be appreciated that many variations,modifications and other applications of the invention may be made.

The invention claimed is:
 1. A medical device comprising a compositiondevoid of cytotoxic or cytostatic activity and comprising a peptidecomprising a sequence selected from the group consisting of YDYNWY (SEQID NO: 1), YDYNLY (SEQ ID NO: 2), FDYNFY (SEQ ID NO: 3), FDYNLY (SEQ IDNO: 4), WDYNLY (SEQ ID NO: 8), FDYNWY (SEQ ID NO: 5), YDWNLY (SEQ ID NO:6) and YDWHLY (SEQ ID NO: 7), wherein the composition is coated on asurface of the medical device or incorporated into a polymeric matrix ofthe medical device, wherein the peptide consists of a length of up to 50amino acids, and wherein the peptide is cyclized.
 2. The medical deviceof claim 1, wherein the medical device is an implantable medical deviceor a medical instrument.
 3. The medical device of claim 2, wherein saidmedical device is selected from the group consisting of artificial bloodvessels, catheters, devices for the removal or delivery of fluids topatients, artificial hearts, artificial kidneys, orthopedic pins,prosthetic joints, plates and implants, urological and biliary tubes,endotracheal tubes, peripherably insertable central venous catheters,dialysis catheters, long term tunneled central venous catheters,peripheral venous catheters, short term central venous catheters,arterial catheters, pulmonary catheters, Swan-Ganz catheters, urinarycatheters, peritoneal catheters, long term urinary devices, tissuebonding urinary devices, artificial urinary sphincters, urinarydilators, ventricular shunts, arterio-venous shunts, breast implants,penile prostheses, vascular grafting prostheses, aneurysm repairdevices, mechanical heart valves, artificial joints, artificiallarynxes, otological implants, anastomotic devices, vascular catheterports, vascular stents, clamps, embolic devices, wound drain tubes,ocular lenses, dental implants, hydrocephalus shunts, pacemakers andimplantable defibrillators, needleless connectors, and voice prostheses.4. The medical device of claim 1, wherein the sequence YDYNWY (SEQ IDNO: 1) is comprised in a protein of sequence LFSVPYDYNWYSNWW (SEQ ID NO:9); or wherein the sequence YDYNLY (SEQ ID NO: 2) is comprised in aprotein of sequence MFSVPYDYNLYSNWV (SEQ ID NO: 58) or LFSVPYDYNLYSNWW(SEQ ID NO: 59); or wherein the sequence FDYNFY (SEQ ID NO: 3) iscomprised in a protein of sequence MFSVPFDYNFYSNWW (SEQ ID NO: 11) orLFSVPFDYNFYSNWW (SEQ ID NO: 59); or wherein the sequence FDYNLY (SEQ IDNO: 4) is comprised in a protein of sequence selected from the groupconsisting of LFSVPFDYNLYSNWW (SEQ ID NO: 18), LFSIPFDYNLYSNWW (SEQ IDNO: 60), MFSVPFDYNLYSNWW (SEQ ID NO: 12), MFSVPFDYNLYTNWW (SEQ ID NO:13), MWSVPFDYNLYSNWW (SEQ ID NO: 14) and MFSVPFDYNLYKNWL (SEQ ID NO:16); or wherein the sequence WDYNLY (SEQ ID NO: 8) is comprised in aprotein of sequence MFSVPWDYNLYKNWF (SEQ ID NO: 15); or wherein thesequence FDYNWY (SEQ ID NO: 5) is comprised in a protein of sequenceMFSVPFDYNWYSNWW (SEQ ID NO: 54); or wherein the sequence YDWNLY (SEQ IDNO: 6) is comprised in a protein of sequence MASIPYDWNLYQSWA (SEQ ID NO:19) or MASIPYDWNLYSAWA (SEQ ID NO: 20); or wherein the sequence YDWHLY(SEQ ID NO: 7) is comprised in a protein of sequence MASIPYDWHLYNAWA(SEQ ID NO: 21).
 5. A device susceptible to biofilm formation,comprising a composition devoid of cytotoxic or cytostatic activity andcomprising a peptide comprising a sequence selected from the groupconsisting of YDYNWY (SEQ ID NO: 1), YDYNLY (SEQ ID NO: 2), FDYNFY (SEQID NO: 3), FDYNLY (SEQ ID NO: 4), WDYNLY (SEQ ID NO: 8), FDYNWY (SEQ IDNO: 5), YDWNLY (SEQ ID NO: 6) and YDWHLY (SEQ ID NO: 7), wherein thepeptide consists of a length of up to 50 amino acids, wherein thecomposition is coated on a surface of the device or incorporated into apolymeric matrix of the device, and wherein the peptide is cyclized. 6.The device of claim 5, wherein the device is selected from the groupconsisting of a vessel hull, an automobile surface, an air planesurface, a membrane, a filter, and an industrial equipment.
 7. Apharmaceutical composition comprising an isolated or synthetic peptidecomprising a sequence selected from the group consisting of YDYNWY (SEQID NO: 1), YDYNLY (SEQ ID NO: 2), FDYNFY (SEQ ID NO: 3), FDYNLY (SEQ IDNO: 4), WDYNLY (SEQ ID NO: 8), FDYNWY (SEQ ID NO: 5), YDWNLY (SEQ ID NO:6) and YDWHLY (SEQ ID NO: 7), wherein the composition is devoid ofcytotoxic or cytostatic activity, wherein the composition comprises apharmaceutically acceptable carrier, wherein the peptide consists of alength of up to 50 amino acids, wherein the pharmaceutically acceptablecarrier enables the composition to be formulated as a pharmaceuticalcomposition adapted for oral, parenteral, rectal, or nasaladministration, and wherein the peptide is cyclized pharmaceuticallyacceptable carrier comprises a stabilizer.
 8. The pharmaceuticalcomposition of claim 7, wherein the sequence YDYNWY (SEQ ID NO: 1) iscomprised in a protein of sequence LFSVPYDYNWYSNWW (SEQ ID NO: 9); orwherein the sequence YDYNLY (SEQ ID NO: 2) is comprised in a protein ofsequence MFSVPYDYNLYSNWV (SEQ ID NO: 58) or LFSVPYDYNLYSNWW (SEQ ID NO:59); or wherein the sequence FDYNFY (SEQ ID NO: 3) is comprised in aprotein of sequence MFSVPFDYNFYSNWW (SEQ ID NO: 11) or LFSVPFDYNFYSNWW(SEQ ID NO: 59); or wherein the sequence FDYNLY (SEQ ID NO: 4) iscomprised in a protein of sequence selected from the group consisting ofLFSVPFDYNLYSNWW (SEQ ID NO: 18), LFSIPFDYNLYSNWW (SEQ ID NO: 60),MFSVPFDYNLYSNWW (SEQ ID NO: 12), MFSVPFDYNLYTNWW (SEQ ID NO: 13),MWSVPFDYNLYSNWW (SEQ ID NO: 14) and MFSVPFDYNLYKNWL (SEQ ID NO: 16); orwherein the sequence WDYNLY (SEQ ID NO: 8) is comprised in a protein ofsequence MFSVPWDYNLYKNWF (SEQ ID NO: 15); or wherein the sequence FDYNWY(SEQ ID NO: 5) is comprised in a protein of sequence MFSVPFDYNWYSNWW(SEQ ID NO: 54); or wherein the sequence YDWNLY (SEQ ID NO: 6) iscomprised in a protein of sequence MASIPYDWNLYQSWA (SEQ ID NO: 19) orMASIPYDWNLYSAWA (SEQ ID NO: 20); or wherein the sequence YDWHLY (SEQ IDNO: 7) is comprised in a protein of sequence MASIPYDWHLYNAWA (SEQ ID NO:21).
 9. The pharmaceutical composition of claim 7, wherein thecomposition is resistant to lyophilization.
 10. The pharmaceuticalcomposition of claim 7, wherein the composition inhibits aggregation ofcells.
 11. The pharmaceutical composition of claim 7, wherein thepharmaceutical composition is selected from one of the following groups:a) tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,or suspensions adapted for oral administration; b) aqueous solutions,oily injection suspensions, water-based injection suspensions adaptedfor parenteral administration; c) suppositories or retention enemasadapted for rectal administration; or d) an aerosol for nasalinhalation.